a) Field of the Invention
The invention is directed to an arrangement for detecting biomolecular reactions and interactions according to the method of reflectometric interference spectroscopy (RS screening method).
b) Description of the Related Art
A process and a device for the detection of physical, chemical, biochemical and biological reactions and interactions are known from DE 196 15 366 A1. In this way, the interactions of biomolecules with biofunctionalized layers in liquid phase are detected in a large number of samples simultaneously by white light interference. Essential parameters for this measurement technique are the detection of the smallest binding detectable with certainty and the stability of this output signal over the binding time span. In the device described herein for carrying out the method, a large number of specimens arranged on a substrate plate or microtiter plate are illuminated with light from a light source via an illumination beam path in which are arranged, among others, a collimator, monochromator and polarizer. The substrate plate or microtiter plate is arranged on a wedge-shaped carrier plate by which a measurement beam path and a reference beam path are generated and imaged on a detector array of a CCD camera via optical elements arranged downstream.
Further, this reference shows that a switchable diaphragm is provided in a focal plane in which the measurement beam path and reference beam path are imaged, wherein these two beam paths can be alternately imaged on the detector array by the switchable diaphragm. In addition, the two above-mentioned beam paths are imaged at the same location on the detector array through a deflecting prism in the reference beam path, so that different sensitivities of the pixel-form receiver elements of the array are referenced out. With this switchable diaphragm, it is only possible to image the measurement beam path and reference beam path on the array at different times; however, short-term variations in light intensity and beam direction of the light source and variations in the spectral transmission characteristic of the monochromator in particular are not eliminated, which could lead to significant measurement uncertainty.
Therefore, it is the primary object of the invention to provide an arrangement for the detection of biomolecular reactions and interactions in which the above-mentioned variations and their effects on measurements are extensively eliminated by means of suitable component assemblies and a suitable referencing concept.
According to the invention, this object is met in an arrangement for the detection of biomolecular reactions and interactions using the RS screening method comprising a specimen plate or microtiter plate for receiving the specimens to be examined, a white light source with subsequently arranged illumination beam path for illuminating the specimens, a splitter plate on which the specimen plate or the microtiter plate is set for generating a detection beam path formed of the measurement beam path and reference beam path, and optical means for imaging the measurement beam path and reference beam path on a spatially resolving detector array of a CCD camera which is connected with an evaluating unit or with a computer for determining measurement values. The arrangement further provides optical imaging elements for imaging the measurement beam path and reference beam path at different positions on the detector array of the CCD camera simultaneously and without overlap, a monochromator which comprises a plurality of interference filters and which is arranged subsequent to the light source in the illumination beam path and a component assembly which collimates the illumination beam path. The component assembly is formed of optical elements and is arranged between the monochromator and the splitter plate and specimen plate or microtiter plate. An optical imaging element is also included for complete spatial separation of the measurement beam path and reference beam path in, or in the vicinity of, an intermediate image plane or aperture diaphragm plane.
Accordingly, it is advantageous when a switchable closure unit is arranged in, or in the vicinity of, the intermediate image plane or aperture diaphragm plane. This makes it possible to record dark references by blocking the measurement beam path and reference beam path and accordingly to eliminate the influence of extraneous light on the measurements.
Further, it is advantageous when the additional optical imaging elements used for separate simultaneous imaging of the measurement beam path and reference beam path are arranged in, or in the vicinity of, the aperture diaphragm plane of the detector beam path comprising these two beam paths.
In accordance with a further embodiment form of the invention, a shared optical imaging element, for example, a lens comprehending both beam paths, is provided for imaging the measurement beam path and reference beam path on the receiver array of the CCD camera. In order to achieve a separation of the measurement beam path and reference beam path in the plane of the detector such that the measurement beam path and reference beam path are free from overlap, at least one deflecting element, each in the form of a deflecting prism, for example, is arranged in front of the optical imaging element in the direction of light in each of these beam paths.
Another advantageous embodiment form of the invention results when the measurement beam path and reference beam path each have their own optical imaging element for spatially separated imaging of these beam paths on the receiver array. This imaging element can be, for example, a lens or also an optical imaging system comprising a plurality of lenses.
According to another feature of the invention, the monochromator which is provided in the illumination beam path subsequent to the polychromatic light source in the direction of light comprises a switchable filter disk in which are arranged a plurality of interference filters. It has turned out in practice that a high detection accuracy can be achieved when working according to the RS method even with a small quantity of spectral channels, so that the evaluation of measurements is significantly simplified. Work can already be carried out successfully with at least three different interference filters, i.e., in three spectral channels. It has proven advantageous to work with six to eight spectral channels, that is, also with an equal number of interference filters. In measurements of changes brought about by the bound specimens in the layer thickness of a suitable polymer film applied to the specimen plate, the spectral channels form the criterion for these measurements to a certain extent. The adjusting accuracy and the stability of the spectral position of the interference extrema and their distance from one another are essential criteria for the selection of the monochromator. Ideally, these criteria are met by interference filter monochromators. In addition, there is the high light-conducting value of these monochromators which is important in the case of large-area illumination of specimen plates or microtiter plates.
In principle, grating monochromators could also be used in the illumination beam path. However, these grating monochromators are disadvantageous, for one, in that they have substantially lower light efficiency compared with interference monochromators. Accordingly, it is also advantageous when a wedge-shaped splitter plate is provided, whose at least one optically active surface has an antireflection coating. With respect to a plane-parallel splitter plate, it is also advantageous when at least one of the optically active surfaces of this splitter plate is provided with an antireflection coating.
When using a plane-parallel plate as beam splitter, the latter must be arranged in the beam path in such a way that it encloses an angle, preferably a small angle, with the specimen plate or microtiter plate carrying the specimens. surface of the plane-parallel plate which is not used for generating the reference beam path is provided with an antireflection coating. The other surface of the plate is advantageously provided with a partially reflecting layer in such a way that a balancing of the reflected intensities in the measurement beam path and reference beam path is achieved over the utilized spectral range.
In another embodiment form of the arrangement according to the invention, the optical elements collimating the illumination beam path and/or the optical imaging elements for imaging the measurement beam path and reference beam path in the aperture diaphragm plane or intermediate image plane and/or the additional optical imaging elements for imaging this aperture diaphragm plane or intermediate image plane on the spatially resolving detector array of the CCD camera are imaging reflectors. It is accordingly advantageous when the optical imaging elements for imaging the measurement beam path and reference beam path in the aperture diaphragm plane or intermediate image plane and/or the additional optical imaging elements for imaging this aperture diaphragm plane or intermediate image plane on the spatially resolving detector array of the CCD camera are convex and concave and/or plane reflectors.
The chromatic aberrations in imaging are minimized to a great extent through the use of reflecting imaging elements such as mirror optics in the form of concave mirrors.
The invention will be described more fully in the following.